Factor VIII (FVIII)
FVIII is a blood plasma glycoprotein of about 280 kDa molecular mass, produced in the liver of mammals. It is a critical component of the cascade of coagulation reactions that lead to blood clotting. Within this cascade is a step in which factor IXa (FIXa), in conjunction with activated factor VIII (FVIIIa), converts factor X (FX) to an activated form, FXa. FVIIIa acts as a cofactor at this step, being required together with calcium ions and phospholipids for maximizing the activity of FIXa. The most common hemophilic disorder is caused by a deficiency of functional FVIII called hemophilia A.
An important advance in the treatment of Hemophilia A has been the isolation of cDNA clones encoding the complete 2,351 amino acid sequence of human FVIII (U.S. Pat. No. 4,757,006) and the provision of the human FVIII gene DNA sequence and recombinant methods for its production).
Analysis of the deduced primary amino acid sequence of human FVIII determined from the cloned cDNA indicates that it is a heterodimer processed from a larger precursor polypeptide. The heterodimer consists of a C-terminal light chain of about 80 kDa in a metal ion-dependent association with an about 200 kDa N-terminal heavy chain. (See review by Kaufman, Transfusion Med. Revs. 6:235 (1992)). Physiological activation of the heterodimer occurs through proteolytic cleavage of the protein chains by thrombin. Thrombin cleaves the heavy chain to a 90 kDa protein, and then to 54 kDa and 44 kDa fragments. Thrombin also cleaves the 80 kDa light chain into a 72 kDa protein. It is the latter protein, and the two heavy chain fragments (54 kDa and 44 kDa above), held together by calcium ions, that constitute active FVIII. Inactivation occurs when the 44 kDa A2 heavy chain fragment dissociates from the molecule or when the 72 kDa and 54 kDa domains are further cleaved by thrombin, activated protein C or FXa. In plasma, FVIII is stabilized by association with a 50-fold molar excess of Von Willebrand Factor protein (“VWF”), which appears to inhibit proteolytic destruction of FVIII as described above.
The amino acid sequence of FVIII is organized into three structural domains: a triplicated A domain of 330 amino acids, a single B domain of 980 amino acids, and a duplicated C domain of 150 amino acids. The B domain has no homology to other proteins and provides 18 of the 25 potential asparagine(N)-linked glycosylation sites of this protein. The B domain has apparently no function in coagulation and can be deleted with the B-domain deleted FVIII molecule still having procoagulatory activity.
Von Willebrand Factor (VWF)
VWF is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in mature plasma VWF (Fischer et al., FEBS Lett. 351: 345-348, 1994). Upon secretion into plasma VWF circulates in the form of various species with different molecular sizes. These VWF molecules consist of oligo- and multimers of the mature subunit of 2050 amino acid residues. VWF can be usually found in plasma as one dimer up to multimers consisting of 50-100 dimers (Ruggeri et al. Thromb. Haemost. 82: 576-584, 1999). The in vivo half-life of human VWF in the human circulation is approximately 12 hours.
The most frequent inherited bleeding disorder in humans is von Willebrand's disease (VWD). Depending on the severity of the bleeding symptoms, VWD can be treated by replacement therapy with concentrates containing VWF, in general derived from human plasma but recombinant VWF also is under development. VWF can be prepared from human plasma as for example described in EP 0503991. In patent EP 0784632 a method for isolating recombinant VWF is described.
VWF is known to stabilize FVIII in vivo and, thus, plays a crucial role to regulate plasma levels of FVIII and as a consequence is a central factor to control primary and secondary hemostasis. It is also known that after intravenous administration of pharmaceutical preparations containing VWF in VWD patients an increase in endogenous FVIII:C to 1 to 3 units per ml in 24 hours can be observed demonstrating the in vivo stabilizing effect of VWF on FVIII.
The patients in general benefit from the specific mode of action of the active ingredients but currently all commercially available Factor VIII preparations are administered via intravenous administration which involves a risk for infections at the injection site and is in general a procedure patients would like to avoid especially in the treatment of children with defects in their coagulation system. Until today the standard treatment of Hemophilia A and VWD involves frequent intravenous infusions of preparations of FVIII and VWF concentrates.
These replacement therapies are generally effective, however, for example in severe hemophilia A patients undergoing prophylactic treatment Factor VIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half life of Factor VIII of about 12 hours. Already by achieving FVIII levels above 1% of normal human plasma corresponding to a raise of FVIII levels by 0.01 U/ml, severe hemophilia A is turned into moderate hemophilia A. In prophylactic therapy the dosing regime is designed such that the trough levels of FVIII activity do not fall below levels of 2-3% of the FVIII activity of non-hemophiliacs.
The administration of a Factor VIII via intravenous administration is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done in home treatment by the patients themselves or by the parents of children being diagnosed for hemophilia A. In addition, frequent intravenous injections inevitably result in scar formation, interfering with future infusions As prophylactic treatment in severe hemophilia is started early in life, with children often being less than 2 years old, it is even more difficult to inject FVIII 3 times per week into the veins of such small patients. For a limited period of time, implantation of port systems may offer an alternative. However, in these cases repeated infections may occur and ports can cause inconvenience during physical exercise.
Thus there is a great medical need to obviate the need to infuse Factor VIII intravenously.
Subcutaneous administration has been proposed for Factor VIII, e.g. in WO 95/01804 A1 and WO 95/026750. However, very high doses of Factor VIII had to be administered to achieve an acceptable bioavailability.
Another approach to improve the bioavailability upon non-intravenous administration has been to use albumin-fused Factor VIII (WO 2011/020866 A2).
WO 2010/077297 A1 and WO 2010/077297 A1 teach the use of hyaluronidase as a spreading or dispersing agent to promote, enhance or increase the dispersion and delivery of a vast number of agents, drugs and proteins to improve the pharmacokinetic and pharmacodynamic profile of the co-administered agent, drug or protein.
It is highly desirable to improve the bioavailability of Factor VIII upon non-intravenous administration. The inventors of this application surprisingly found that the bioavailability of Factor VIII is substantially increased if it is administered in combination with a sulfated glycosaminoglycan and a hyaluronidase.